Furin polypeptides with improved characteristics

ABSTRACT

The present invention comprises a furin polypeptide having a modified amino acid sequence between the middle, homo-B-domain and the transmembrane domain compared to wild-type furin which retains proteolytic activity but is secreted at lower levels in cell culture compared to wild-type furin. Additionally, the invention includes nucleic acid molecules encoding such furin polypeptides, vectors and host cells comprising said nucleic acid molecules, compositions comprising said furin polypeptide and methods for producing such compositions.

BACKGROUND OF THE INVENTION

The present invention relates to new furin polypeptides. Furin, alsocalled PACE (for paired basic amino acid cleavage enzyme), belongs tothe family of mammalian subtilisin-like proprotein convertases (SPC orPC) These proteins have been implicated in the endoproteolyticmaturation processing of inactive precursor proteins at single, pairedor multiple basic consensus sites within the secretory pathway (reviewedin Nakayama, 1997, Biochem.J., 327, pp. 625-635; (Seidah and Chretien,Current Opinions in Biotechnology,8, 1997, pp. 602-607). Seven distinctmembers of this family have been identified to date, including furin,PC1 (also known as PC3), PC2, PACE4, PC4, PC5 (also known as PC6), PC7(or LPC, PC8, or SPC7), each of which exhibits unique tissuedistribution, although overlapping functional redundancy of various PCsin some tissues may occur (Seidah et al., Biochem.,1994, 76, pp.197-209).

Furin is ubiquitously expressed in all mammalian tissues and cell lineswhich have been examined, and is capable of processing a wide range ofbioactive precursor proteins in the secretory pathway, including growthfactors, hormones, plasma proteins, receptors, viral envelopeglycoproteins and bacterial toxins. It is a calcium-dependent serineendoprotease structurally arranged into several domains, namely a signalpeptide, propeptide, catalytic domain, middle domain, (also termedhomo-B or P-domain), the C-terminally located cysteine-rich domain,transmembrane domain and the cytoplasmic tail. Upon transit of the newlysynthesized furin precursor from the endoplasmic reticulum to the Golgicompartment, the propeptide is autocatalytically removed in a two stepprocessing event (Leduc et al., J.Biol.Chem., 267, 1992, pp.14304-14308; Anderson et al., EMBO J., 1997, pp. 1508-1518).

Furin is predominantly localized to the trans-Golgi network (TGN), butit also cycles between the TGN and the cell surface via endosomalvesicles, thereby processing both precursor proteins during theirtransport through the constitutive secretory pathway as well asmolecules entering the endocytic pathway. The cellular distribution offurin to the varied processing compartments is apparently directed bydefined structural features within its cytoplasmic tail (Schäfer et al.,EMBO J.,11, 1995, pp. 2424-2435; Voorhees et al., EMBO J., 20, 1995, pp.4961-4975; Teuchert et al., J.Biol.Chem., 274, 1999, pp. 8199-8207).Deletion of the cytoplasmic domain results in a truncated furinpolypeptide located primarily in the plasma membrane, to which it istransported probably by a default pathway, incapable of recycling to theTGN due to the loss of regulative sequence motifs within the cytoplasmicdomain (Molloy et al., EMBO J., 13, 1994, pp. 18-33; Schäfer et al.,EMBO J., 14, 1995, pp. 2424-2435).

The C-terminal domains have been found to be dispensable for thefunctional activity of furin. Mutant furin lacking the transmembranedomain and the cytoplasmic tail, was found to be readily released intocell culture medium while still exhibiting significant activity. Highlevels of expression of full length recombinant furin have resulted inthe natural secretion of a truncated furin form, called ‘shed’ furin,which lacks the transmembrane domain and the cytoplasmic tail (Wise etal., Proc.Natl.Acad.Sci., 87, 1990, pp. 9378-9382; Rehemtulla andKaufman, Blood, 79, 1992, pp. 2349-2355; Vidricaire et al.,Biochem.Biophys.Res.Comm., 195, 1993 pp. 1011-1018; Vey et al.,J.Cell.Biol., 127, 1994, pp. 1829-1842; Preininger et al.,Cytotechnol.,30, 1999, pp. 1-15). It remains an open question as to whether furinshedding is due to saturating cellular retrieval mechanisms, whether itrepresents a protection mechanism of the host cell against excessprotease, or whether is part of a natural regulatory process modulatingintracellular furin concentration/activity by secretion. The isolationof a truncated endogenous furin from the Golgi fraction of bovine kidneycells may support the view that shedding is not solely an artificialsecretion process caused by overexpression (Vey et al., 1994).Conversion of furin into the soluble secreted form was shown to occurintracellularly within an acidic compartment which requires the presenceof calcium (Vey et al., 1994).

The presence of a C-terminal truncated and hence soluble form of furinthat remains active, however, has been detected almost exclusively inconditioned medium of cells recombinantly overexpressing nativefull-length furin (Wise et al., 1990; Rehemtulla and Kaufman, 1992;Vidricaire et al., 1993; Vey et al., 1994; Preininger et al., 1999).

Other prior art describing furin polypeptides includes WO 91/06314,which describes a fragment of furin consisting of amino acids 108-464,thus lacking part of the homo-B domain, the cysteine-rich region, thetransmembrane domain and the cytoplasmic tail. WO 92/09698 disclosesfull length furin and furin lacking the transmembrane domain. Inaddition, Preininger et al. (Cytotechnology 30, 1999, pp. 1-15) describefurin mutants lacking the cysteine rich region, the trans-membranedomain and the cytosolic domain. Cells expressing such mutants containedincreased intracellular concentrations of the furin derivatives butvarying levels of secretion. The authors stated that the lack ofextracellular accumulation of these molecules suggested that thesemolecules were most likely degraded. The authors stated further thatfull length recombinant furin, located intracellularly, seems to belargely inactive and that there is a potential toxicity of largeramounts of full length furin to its host cell.

SUMMARY OF THE INVENTION

We have found that soluble furin in a cell culture medium can causeproteins which are not naturally processed by furin to be unspecificallycleaved. For example, although native Factor VIII is not naturallyprocessed by furin, Factor VIII can become a target for inadvertantprocessing by soluble furin when exposed to furin for an extended periodof time, e.g. in a cell culture medium. This leads to, a reduced yieldof structurally intact Factor VIII protein in such cell culture medium.This can be the case when Factor VIII is coexpressed together with anatural substrate of furin, e.g. von Willebrand Factor, or whenrecombinant proteins which are naturally processed by furin are exposedto furin for an extended period of time so that in addition inadvertentsites are cleaved.

The present invention reduces or prevents unspecific cleavage ofproteins in cell culture through the use of modified furin polypeptideswhich have proteolytic activity but which are not secreted into culturemedium by host cells or are secreted in reduced amounts compared to thesecretion of wild-type furin. Such furin polypeptides have been foundnot to be toxic to host cells even when expressed intracellularly inhigh amounts.

Accordingly, the present invention provides a furin polypeptide having amodified amino acid sequence compared to that of wild-type furin betweenhomo-B-domain and the transmembrane domain, that is, between amino acidsAla 557 and Leu 713 according to the amino acid sequence presented inFIGS. 1 and 2. It has been surprisingly found that furin polypeptideshaving such a modified amino acid sequence have proteolytic activitysimilar to that of native (i.e., wild-type) furin, but are secreted byhost cells expressing such furin polypeptides into cultivation medium insubstantially reduced amounts compared to native furin.

It is another aspect of the invention that the furin polypeptidesaccording to the invention can be expressed in high amounts in a cellwithout being substantially toxic to the cell. In still a furtheraspect, the physiological cleavage properties of the modified furinprotein are still present, but inadvertent cleavage of secreted orextracellularly localized proteins in a cell culture medium is highlyreduced since less or no furin is present in the medium.

Additionally, a further advantage of the furin polypeptide of thepresent invention is that although the proteolytic processing offurin-dependent proteins can occur intracellularly, unspecificprocessing of proteins by furin can be at least reduced if notcompletely eliminated. Therefore, unspecific cleavage of proteins whichmight occur when proteins are exposed to soluble furin in a conditionedmedium in cell culture is avoided by the furin polypeptide according tothe present invention.

In another aspect, the invention provides a recombinant polynucleotideencoding the furin polypeptide according to the present invention. Inyet another aspect, the invention provides a method for producing thefurin polypeptide according to the present invention, a recombinantvector comprising the polynucleotide sequence encoding the furinpolypeptide according to the invention, a host cell comprising suchvector, and a preparation comprising the furin polypeptide of thepresent invention.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of human wild-type furin (SEQ IDNO:1).

FIG. 2 is a schematic representation of the amino acid sequences ofwild-type furin and furin mutants.

FIG. 3 is a photograph of an SDS-PAGE gel showing shed furin in aconditioned medium in which FD11-CHO-rvWF cells transiently transfectedwith furin constructs were grown.

FIG. 4 is a photograph of an SDS-PAGE gel showing the processing of rvWFprecursor in FD11-CHO-rvWF cells transiently transfected with furinconstructs.

FIGS. 5A-5C show furin expression in transiently transfected HEK 293cells:

FIG. 5A is a photograph of an SDS-PAGE gel showing shed recombinantfurin (rfurin) in conditioned medium of transiently transfected HEK 293cells;

FIG. 5B is a photograph of an SDS-PAGE gel showing intracellular rfurinexpression in HEK 293 lysates; and

FIG. 5C shows the results of an in vitro furin assay using conditionedmedium and fluorogenic substrate (in arbitrary units).

FIG. 6 is a photograph of three SDS-PAGE gels showing the correlationbetween the degree of rvWF precursor processing and the presence of shedfurin in conditioned medium.

DETAILED DESCRIPTION OF THE INVENTION

Furin Polypeptides

The present invention comprises furin polypeptides which have a modifiedamino acid sequence between amino acids Ala 557 and Leu 713 compared tothe amino acid sequence of wild-type mammalian furin, such as humanfurin (the amino acid sequence of which is shown in FIG. 1). Forpurposes of the present disclosure, a furin polypeptide shall refer to apolypeptide comprising at least a portion of the amino acid sequence ofa wild-type mammalian furin protein which has proteolytic activity. In apreferred embodiment the modification in a furin polypeptide accordingto the present invention is located between amino acids Ala 557 and Leu713. In an alternative embodiment, the modification is at Arg 683. Instill another embodiment, the amino acids between Gly 577 and His712 aredeleted.

In the instant disclosure, the terms “modified” and “modification” shallmean, with respect to the amino acid sequence of a furin polypeptide, anaddition, deletion or substitution of one or more amino acids. Such amodification can be carried out by, for instance, directed mutagenesisor PCR or other methods of genetic engineering known in the art whichare suitable for specifically changing a DNA sequence in order to directa change in the amino acid sequence of the resulting polypeptide(Current Protocols in Molecular Biology, vol. 1, ch. 8 (Ausubel et al.eds., J. Wiley and Sons, 1989 & Supp. 1990-93); Protein Engineering(Oxender & Fox eds., A. Liss, Inc., 1987). The modifications of thepresent invention are in the region between the homo-B-domain and thetransmembrane domain, i.e. the region between the amino acids Ala 557and Leu 713, of the furin molecule.

Preferably, the furin polypeptide of the present invention has aminoacid substitutions and/or additions creating loop or alpha-helixstructures. It is well known from the prior art that amino acids canform several different secondary structures in polypeptides, i.e.helical or looped structures (Lehninger A., “Biochemie”, VCH, 1985, pp.102-107; Karlson P. et al., “Kurzes Lehrbuch der Biochemie, Georg ThiemeVerlag, 1994; pp. 29-32). These structures can be produced by selectingspecific amino acids which form, for example, alpha helices and loopsand thereby developing structures like helices or loops in the resultingpolypeptide (Rost B. and Sander C., Proc.Natl.Acad.Sci., 1993, pp.7558-7562, Rost B. and Sander C., 1994, Proteins: Structure, Functionand Genetics, 19, pp. 55-72). Additionally, according to Kyte J. andDoolittle R. (1983, J.Mol.Biol., 157, pp. 105-132) such amino acids maybe selected based on their hydropathy values, in view of the knowledgethat amino acids showing negative hydropathy values are hydrophilic,allowing these side chains access to the aqueous solvent, whereas aminoacids showing positive hydropathy values are hydrophobic amino acidswhich tend to comprise interior portions of the proteins. Additionally,it is known that amino acids showing very high positive or negativehydropathy values are preferred targets for various proteases.

Therefore, in a preferred embodiment, there is an insertion of severalamino acids, preferably between 5 and 30, more preferably between 10 to20, which produce a loop or helix structure in the modified furinpolypeptide of the present invention.

In an alternative embodiment, the insertion of amino acids results in ahelix structure. In such an embodiment the amino acids are preferablyselected from the group consisting of alanine (A), leucine (L),phenylalanine (F), tryptophan (W), methionine (M), histidine (H),glutamine (Q), valine (V) and glutamic acid (E). For example, the aminoacids 558 to 738, preferably amino acids 578 to 711 are substituted bythe amino acid sequences EAMHA (SEQ ID NO:2), AWFQW (SEQ ID NO:3) ORAQMWHEAMEFWAMQFEAMHA (SEQ ID NO:4). In a preferred embodiment, aminoacids 578 to 711 of the furin polypeptide are substituted by the aminoacid sequence AEMWHQAMEV (SEQ ID NO:5).

In yet another embodiment, an amino acid insertion builds up a loopstructure, wherein the amino acids are preferably selected from thegroup consisting of serine (S), isoleucine (I), threonine (T), glutamicacid (E), aspartic acid (D), lysine (K), arginine(R), glycine (G),tyrosine (Y), cysteine(C), asparagine (N), proline (P), glutamine (Q)and hydroxyproline. For example, the amino acids 558 to 738, preferablyamino acids 578 to 711 are substituted by the amino acid sequences SYNPG(SEQ ID NO:6), SYQPD (SEQ ID NO:7) or GSPYQTNGPS (SEQ ID NO:8). In apreferred embodiment, amino acids 578 to 711 of the furin polypeptideare substituted by the amino acid sequence GSPNSQPYDG (SEQ ID NO:9).

The selection of amino acids for forming looped and helical structuresis well known to the skilled person (Lehninger A., “Biochemie”, VCH,1985, pp. 102-107).

In an alternative embodiment, the arginine at amino acid position 683 ofthe furin sequence can be replaced by any of the amino acids, preferablyby lysine, glutamic acid or isoleucine.

Nucleic Acids and Vectors

Another embodiment of the invention provides polynucleotides whichencode the furin polypeptides of the present invention. The nucleicacids used in such polynucleotides may be DNA and/or RNA.

A full-length furin polynucleotide as well as any derivatives thereofencoding a furin polypeptide having proteolytic activity can be used asthe starting material for the construction of the furin polypeptides ofthe present invention. The cDNA sequence encoding native human furin waspublished by van den Ouweland, A. M. W. et al. (Nucleic Acid Res., 1990,18(3), p. 664) and Fuller R. S. et al. (Science, 1989, 246:482). Such afurin polynucleotide can originate from any mammalian species,preferably from human, porcine or bovine sources.

The polynucleotide is expressed by a vector that provides theappropriate elements for the heterologous expression of said DNA or RNA.The expression vector comprises, for example, a transcriptionalregulatory region and a translational initiation region functional in ahost cell, a DNA sequence encoding for the furin polynucleotide of thepresent invention and translational and transcriptional terminationregions functional in said host cell, wherein expression of said nucleicsequence is regulated by said initiation and termination regions.

The expression vector may also contain elements for the replication ofsaid DNA or RNA. The expression vector may be a DNA or an RNA vector.Examples for DNA cloning and expression vectors are pBSSKII (Short, J.M., Fernandez, J. M., Sorge, J. A. and Huse, W. D. Lambda ZAP, 1988,Nucleic Acids Research 16 (15), 7583-7600; Alting. Mees, M. A., andShort, J. M., 1989, Nucleic Acids Research 17 (22), 9494), pBPV, pSVL,pCMV, pRc/RSV, myogenic vector systems (WO 93/09236) or vectors derivedfrom viral systems, for example from vaccinia virus, adenoviruses,adeno-associated virus, herpesviruses, retroviruses or baculoviruses.Examples for RNA expression vectors are vectors derived from RNA viruseslike retroviruses or flaviviruses.

In some instances it might be desirable to have a plurality of copies ofthe gene expressing the protein precursor in relation to the furinpolypeptide, or vice versa. This can be achieved in ways well describedin the prior art. Alternatively, one can employ two transcriptionalregulatory regions having different rates of transcriptional initiationor different promoters, providing for enhanced expression of either thefurin polypeptide according to the invention or the expression of theprecursor polypeptide and/or a further polypeptide which is not to beproteolytically processed by furin.

The expression vector containing the polynucleotide which encodes themodified furin polypeptide according to the present invention can beused to transform host cells which then produce said polypeptide. Thetransformed host cells can be grown in a cell culture system to producesaid polypeptide in vitro.

For some specific applications in gene therapy, i. e. when the nucleicacid per se is injected into an organ of a mammal, the nucleic acid, DNAas well as RNA, may be chemically modified. The chemical modificationsmay be modifications that protect the nucleic acid from nucleasedigestion, for example by stabilizing the backbone or the termini.

The expression vector containing the nucleic acid which encodes a furinpolypeptide of the present invention can further be administered to amammal without prior in vitro transformation into host cells. Thepractical background for this type of gene therapy is disclosed inseveral patent applications, for example in WO 90/11092. The expressionvector containing said nucleic acid is mixed with an appropriatecarrier, for example a physiological buffer solution, and is injectedinto an organ, preferably skeletal muscle, the skin or the liver of amammal.

Host Cells

The modified furin polypeptide according to the present invention ispreferably produced by recombinant expression. It can be prepared bymeans of genetic engineering with expression systems known to the art,such as, for instance, permanent cell lines or viral expression systems.Permanent cell lines are prepared by stable integration of theextraneous DNA into the host cell genome of, e.g., vero, MRC5, CHO, BHK,293, HEK 293, Sk-Hep1, liver cells, kidney cells, fibroblasts,keratinocytes or myoblasts, hepatocytes or stem cells, for examplehematopoietic stem cells, or by an episomal vector derived, for example,from papilloma virus.

Alternatively, cell lines having no endogenous furin activity can beused (Moehring J. M. and Moehring T. J., Infect.Immun., 41, 1983, pp.998-1009). For example, CHO-RPE40 or FD11-CHO-cells can be used.Therein, the proteolytic activity of the transfected furin of theinvention can be easily measured, avoiding the background activity ofendogenous furin.

Viral expression systems, such as, for instance, the vaccinia virus,baculovirus or retroviral systems, can also be employed. As cell lines,vero, MRC5, CHO, BHK, 293, Sk-Hep1, gland, liver or kidney cells aregenerally used. Eukaryotic expression systems, such as yeasts,endogenous glands (e.g. glands of transgenic animals) and transgenicanimals can also be used for the expression of the furin polypeptidesaccording to the present invention. For the expression of recombinantproteins, CHO-DHFR-cells have proved particularly useful (Urlaub et al.,Proc.Natl.Acad.Sci., USA, vol 77, pp. 4216-4220, 1980).

The furin polypeptides according to the present invention are expressedin the respective expression systems under the control of suitablepromoters. For expression in eukaryotes, known promoters are suitable,such as SV40, CMV, RSV, HSV, EBV, β-actin, hGH or inducible promoterssuch as hsp or metallothionein promoter.

In a preferred embodiment the present invention provides a method forthe production of a furin polypeptide according to the present inventionand a precursor polypeptide. Preferably, the furin polypeptide iscoexpressed with von Willebrand factor protein and/or Factor VIIIprotein.

In a further aspect the invention provides a method for the productionof a furin polypeptide according to the present invention. This methodcomprises growing in a nutrient medium a host cell comprising anexpression vector which comprises, in the direction of transcription, atranscriptional regulatory region and a translational initiation regionfunctional in a host cell, a DNA sequence encoding a furin polypeptideof the invention, and translational and transcriptional terminationregions functional in said host cell. The expression of this DNAsequence is regulated by the initiation and termination regions. Themethod can further include measuring the secretion rate of expressedfurin polypeptides with proteolytic activity and isolating host cellsexpressing furin polypeptides showing reduced secretion compared to hostcells expressing wild-type furin.

Pharmaceutical Preparation

The furin polypeptide according to the present invention can be providedas a pharmaceutical preparation having a modified furin polypeptideaccording to the present invention as a single component preparation orin combination with other components as a multiple component system. Ina particular embodiment, a furin polypeptide of the invention can becombined with pro-proteins, for example von Willebrand Factor.

Specific Activity

According to one aspect of the present invention, the furin polypeptideof the invention has a furin proteolytic activity of at least 50%,preferably at least 100% compared to the proteolytic activity ofwild-type furin protein, such as wild-type human furin.

The evaluation of proteolytic activity can be performed by any suitabletest, for example by using fluorogenic substrates which are comprised ofa dibasic cleavage site for which furin is specific (Preininger A. etal., 1999, Schlokat U. et al., 1996, Biotechnol. Appl. Biochem., vol.24, pp. 257-267). Alternatively the proteolytic activity can also bemeasured by incubating furin with pro-proteins, for example pro-rvWF,for a sufficient time. The degree of pro-rvWF processing can be analysedby Western blotting.

Secretion Rate

The secretion rate can be defined as the amount of secreted furinpolypeptide (shed furin) which accumulates in a cell culture mediumwithin a given time. The reduction in the secretion rate of the modifiedfurin polypeptide according to the present invention is at least 25%,preferably at least 50%, more preferably at least 90%, most preferably100% compared to the secretion rate of recombinantly expressed furinhaving the wild-type sequence (such as wild-type human furin) or furinlacking the transmembrane and/or cytoplasmic region.

For example, the secretion rate can be measured by immunologicalreactivity with anti-furin antibodies. A suitable antibody can bedirected against the catalytic domain of furin (Preininger et al., 1999)

Isolation Methods

The furin polypeptide according to the present invention can be isolatedfrom cells by lysis and further purified by conventional methods,optionally in the presence of protease inhibitors. The purification canbe done by chromatographic methods known in the art, preferably byaffinity chromatography, using antibodies against the furin polypeptideor by coupling the furin polypeptide to a His-Tag group and selectivelybinding the protein on Ni²⁺-NTA agarose (Preininger et al., 1999)

Due to the fact that the proteolytic characteristics of the furinpolypeptides of the present invention compared to wild type furin aresubstantially unaltered, proteins that are processed by wild-type furincan also be processed by the furin polypeptides of the invention, i.e.proteins with paired amino acid residues can serve as a substrate.Examples of precursor molecules for use in the present invention caninclude, but are not limited to, von Willebrand Factor; Factor IX,protein C, protein S, prothrombin, Factor X, Factor VII, transforminggrowth factor (TGF) beta and its superfamily, including activin andinhibin, bone morphogenetic proteins (BMP), insulin, relaxin, growthfactors like platelet derived growth factor (PDGF), nerve growth factor(NGF), and virus polypeptides including those from cytomegalovirus(CMV), human immunodeficiency virus and herpes simplex virus.

The invention is illustrated in the subsequently described examples.Variations within the purview of one skilled in the art are to beconsidered to fall within the scope of the present invention. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. The following examples illustrate thepresent invention but do not limit the scope of the invention in anyway.

EXAMPLES

1. Construction of Furin R683A

Full length furin mutant R683A, harboring the amino acid alanine insteadof the native arginine at position 683, was constructed using aPCR-based approach with overlapping extended primers (Ho et al., 1989,Gene, 77, pp. 51-59). Initially, two standard PCR reactions wereperformed using plasmid pCMV-furin wt (harboring the furin wild-typecDNA) as template and primer pairs 4953 (5′ GGGGGATCCCTCTGGCGAGTGG 3′)(SEQ ID NO:10) and 5210 (5′ CGGGGACTCTGCGCTGCTCTG 3′) (SEQ ID NO:11) or5209 (5′ CAGAGCAGCGCAGAGTCCCCG 3′) (SEQ ID NO: 12) and 4954 (5′GGGGGATCCCCGCGGCCTAGG 3′) (SEQ ID NO: 13), where 5210 and 5209 are theinner complementary extended primers introducing the mutation, and 4953and 4954 are the outer primers containing a Bam HI restriction site. Ina second PCR round, the two purified amplification products of theinitial PCR reactions were combined for overlap extension in thepresence of the two outer primers 4953 and 4954. The final purified PCRproduct was digested with Bam HI and was used to replace the wild-typeBam HI fragment in plasmid pCMV-furin wt.

2. Construction of Furin Deletion Mutants Helix 10, Loop 10 and 578-711

Furin expression constructs Helix 10 (comprising a deletion of aminoacid residues 578-711 replaced by 10 helical structured residues), Loop10 (comprising a deletion of amino acid residues 578-711 replaced by 10loop structured residues) and 578-711 (comprising a deletion of aminoacid residues 578-711) were generated by inverse PCR. For that purpose,the internal 1176 bp Bam HI fragment of wild-type furin was subclonedinto the Bam HI site of vector pBS SKII(+) (Stratagene). The resultingplasmid pBS/fur1176 was used as the template for the inverse PCRreactions of the individual constructs. In the case of Helix 10 and Loop10, the specific sense and reverse primers each contained at their5′-end an additional overhanging 15 nucleotides coding for 5 helicalor.loop structured amino acids. The following primer sets were used: forHelix 10, sense primer 5699 (5′CAGGCCATGGAGGTGCACCTGCCTGAGGTGGTGGCCGGCCTCAGC 3′) (SEQ ID NO.14) andreverse primer 5700 (5′ GTGCCACATCTCGGCCCCCTCAGGGGCGGTGCCATAGAGTACGAG 3′(SEQ ID NO:15), for Loop 10, sense primer 5701 (5′CAGCCCTACGACGGCCACCTGCCTGAGGTGGTGGCCGGCCTCAGC 3′) (SEQ ID NO:16) andreverse primer 5702 (5′ GCTGTTGGGGCTGCCCCCCTCAGGGGCGGTGCCATAGAGTACGAG3′) (SEQ ID NO:17), and for 578-711, sense primer 5723 (5′CACCTGCCTGAGGTGGTGGCC 3′) (SEQ ID NO:18) and reverse primer 5724 (5′CCCCTCAGGGGCGGTGCCATA 3′) (SEQ ID NO:19). The resulting PCR-fragmentswere purified, treated with T4 polynucleotide kinase (New EnglandBiolabs), religated with T4 DNA-ligase (Roche) and transformed into E.coli strain XL1 Blue MRF′ (Stratagene). Positive clones, harboring theintroduced mutation were selected by sequencing, and the mutated BamHIfragment was used to replace the wt 1176bp BamHI fragment in pCMV-furinwt.

Generally, amplification of the target sequences was routinely carriedout within 30 PCR cycles using 10-20 ng template DNA in a total volumeof 100 μl containing 30 pMol of each primer, 200 μM of each dNTP, 2 mMMgSO₄ in the supplied 10×PCR buffer and 2.5 U Vent_(R)® DNA polymerase(New England Biolabs) at 55° C. annealing and 72° C. extensiontemperatures. PCR-fragments were purified using QIAEX II Gel ExtractionKit (Qiagen) according to the supplier's instructions.

The Helix 10 insertion into the furin deletion mutant Δ578-711 comprisesthe amino acid sequence AEMWHQAMEV (SEQ ID NO:20). The Loop 10 insertioninto the furin deletion mutant Δ578-711 comprises the amino acidsequence GSPNSQPYDG (SEQ ID NO:21).

3. Transfection, Cell Culture and Protein Harvest

Furin constructs were transiently expressed in 293 HEK (human embryonickidney fibroblasts; ATCC CRL 1573) and FD11-CHO-rvWF cells (FD11-CHO arefurin deficient cells). The cells were grown in DMEM/Ham's F12 (1:1)medium (Life Technologies) supplemented with 10% fetal calf serum (fullmedium) . For transfection, cells were grown to 50-75% confluency on 5cm culture dishes (Costar) and transfected by calcium phosphatecoprecipitation as described previously (Fischer et al., 1994).Transient transfections were carried out with 20 μg of expressionplasmid.

Recombinant protein was harvested by applying serum-free full medium tothe transfected cells upon confluency (generally 48 hourspost-transfection), after washing them twice with PBS (Ca²⁺ and Mg²⁺free, Life Technologies). Conditioned medium was collected and clearedby centrifugation. Adherent cells were trypsinized, washed with PBS andthe total cell number was determined by a CASY counter (Schärfe Systems,Germany) employing a 30 μm capillary. Cell extracts were prepared bylysing the cells at a concentration of 5×10⁷ cells/ml lysis buffer,containing 20 mM Tris-HCl, pH7.5, 150 mM NaCl, 1 mM EDTA and 0.5%Tritone® X-100. After incubation for 30 min at 4° C., lysates werecleared by centrifugation for 15 min at 10,000×g at 4° C.

4. Western Blotting

Samples were reduced and denatured, resolved by SDS-PAGE on 4%stacking/8% or 10% separation gels, and visualized by Western blottingas described (Schlokat et al., 1996). Conditioned medium derived fromFD11-CHO-rvWF transient transfections was concentrated 20× by speed-vaccentrifugation prior to loading. Lysates were applied per slot onSDS-PAGE equivalent to 7.5×10 cells. For the detection of furinmolecules, murine monoclonal antibody MON-148 (Alexis) directed againstthe catalytic domain of furin and alkaline phosphatase conjugated toanti-mouse IgG goat sera (Sigma) as the second antibody was used.Recombinant vWF was visualized employing rabbit anti-vWF antiserum(DAKO) and alkaline phosphatase conjugated to anti-rabbit IgG goat sera(Promega) as the second antibody.

FIG. 3 shows the amount of shed furin in conditioned medium oftransiently transfected FD11-CHO-rvWF cells. The conditioned medium wasconcentrated 20× and applied and denatured on 4% stacking/10% separationSDS-PAGE gel. The Western blot was visualized with MON-148 andAP-conjugated anti-mouse IgG antibody.

As a control, a pCMV vector, wild-type furin polypeptide andΔ577G-4×G-10×H were used. The furin construct Δ577G-4×G-10×H wasprepared according to Preininger et al. (1999).

The figure clearly shows that the furin constructs according to theinvention do not show any shedding, i.e. the secretion rate of themolecules into the medium is substantially reduced compared to rfurinhaving the wild-type sequence or furin lacking the transmembrane andcytoplasmic domains.

5. Analysis of in vitro Furin Activity in Conditioned Medium

Functional activity of shed furin molecules was determined byfluorogenic substrate as described previously (Schlokat et al., 1996).

6. Evidence of Intracellular rfurin Activity

FD11-CHO-rvWF cells stably expressing furin mutant R683A or wild-typefurin were established by cotransfection using 20 μg furin expressionplasmid and 1 μg selection plasmid pCMV-hyg mediating resistance tohygromycin B (Roche). Resistant clones were isolated two weeks aftertransfection and stabilized by subcloning under selective pressure.Three FD11-CHO-rvWF/R683A clones (clone 1, 2 and 3) differing in theamount of secreted rfurin and consequently showing variable degrees ofrvWF precursor processing were selected.

Intracellular furin activity was demonstrated by correlating the degreeof rvWF precursor processing and the presence of shed rfurin inFD11-CHO-rvWF/R683A conditioned media over a time period of 24 hours. Ascontrols, FD11-CHO-rvWF/furin wt and FD11-CHO-rvWF cells were used.Cells were grown in 6-well dishes (one well/timepoint) until confluency,and washed two times with PBS before serum-free medium was applied for atime period of 4, 8, 16 and 24 hours. Conditioned medium was cleared bycentrifugation and concentrated 20× for the detection of shed rfurin.Estimation of rvWF precursor processing was done by Western blot.

FIG. 4 shows the processing of rvWF precursor in transiently transfectedFD11-CHO-rvWF cells. 100 ng rvWF was applied per lane. Probes werereduced denatured and applied on 4% stacking/5% separating SDS-PAGE gel.The Western blot was developed with polyclonal rabbit-anti-vWF andAP-conjugated anti-rabbit IgG antibody. Although the cells were onlytransiently transfected, the R683A, Helix 10, Loop 10 and Δ578-711 furinconstructs evidence proteolytic activity. The term transientlytransfected reflects a genetically non-homogenous, mixed cellpopulation. Depending on the transfection efficiency, only some of thecells are transfected.

FIG. 5 (comprising FIGS. 5A-5C) shows furin expression in transientlytransfected HEK293 cells:

FIG. 5A shows shed furin in conditioned medium of transientlytransfected HEK293 cells. 15 μl of conditioned medium were applied perslot. Probes were reduced and denatured and applied on 4% stacking/10%separating SDS-PAGE. The Western blot was developed with MON-148 andAP-conjugated anti-mouse IgG antibody.

FIG. 5B shows the measurement of intracellular rfurin in HEK293 lysates.7.5×10e5 cell equivalents were applied per slot.

FIG. 5C shows the results of an in vitro assay using conditioned mediumand a fluorogenic substrate.

FIG. 5A shows that the amount of secreted furin polypeptides in themedium detectable by a specific antibody is highly reduced. This isconfirmed by the in vitro activity measurements shown in FIG. 5C. Thedata of FIG. 5B show that the furin polypeptides are locatedintracellularly.

FIG. 6 shows the intracellular proteolytic activity of the furinconstruct R683A. The degree of rvWF precursor protein processing and thepresence of shed rfurin in the conditioned medium is compared. Thefigure shows that significant proteolytic processing of vWF proteinoccurs even though no shed furin is detected in the medium. Thisindicates that this furin polypeptide is proteolytically active eventhough it is not secreted into the medium.

The upper lane is a vWF western blot, wherein 100 ng rvWF is applied perlane. As a positive control, CHO-rvWf was used.

The lower lane is a furin western blot of conditioned medium. Thematerial was concentrated 20× per lane. As a positive control, shedwild-type rvWF was used.

21 1 794 PRT Human 1 Met Glu Leu Arg Pro Trp Leu Leu Trp Val Val Ala AlaThr Gly Thr 1 5 10 15 Leu Val Leu Leu Ala Ala Asp Ala Gln Gly Gln LysVal Phe Thr Asn 20 25 30 Thr Trp Ala Val Arg Ile Pro Gly Gly Pro Ala ValAla Asn Ser Val 35 40 45 Ala Arg Lys His Gly Phe Leu Asn Leu Gly Gln IlePhe Gly Asp Tyr 50 55 60 Tyr His Phe Trp His Arg Gly Val Thr Lys Arg SerLeu Ser Pro His 65 70 75 80 Arg Pro Arg His Ser Arg Leu Gln Arg Glu ProGln Val Gln Trp Leu 85 90 95 Glu Gln Gln Val Ala Lys Arg Arg Thr Lys ArgAsp Val Tyr Gln Glu 100 105 110 Pro Thr Asp Pro Lys Phe Pro Gln Gln TrpTyr Leu Ser Gly Val Thr 115 120 125 Gln Arg Asp Leu Asn Val Lys Ala AlaTrp Ala Gln Gly Tyr Thr Gly 130 135 140 His Gly Ile Val Val Ser Ile LeuAsp Asp Gly Ile Glu Lys Asn His 145 150 155 160 Pro Asp Leu Ala Gly AsnTyr Asp Pro Gly Ala Ser Phe Asp Val Asn 165 170 175 Asp Gln Asp Pro AspPro Gln Pro Arg Tyr Thr Gln Met Asn Asp Asn 180 185 190 Arg His Gly ThrArg Cys Ala Gly Glu Val Ala Ala Val Ala Asn Asn 195 200 205 Gly Val CysGly Val Gly Val Ala Tyr Asn Ala Arg Ile Gly Gly Val 210 215 220 Arg MetLeu Asp Gly Glu Val Thr Asp Ala Val Glu Ala Arg Ser Leu 225 230 235 240Gly Leu Asn Pro Asn His Ile His Ile Tyr Ser Ala Ser Trp Gly Pro 245 250255 Glu Asp Asp Gly Lys Thr Val Asp Gly Pro Ala Arg Leu Ala Glu Glu 260265 270 Ala Phe Phe Arg Gly Val Ser Gln Gly Arg Gly Gly Leu Gly Ser Ile275 280 285 Phe Val Trp Ala Ser Gly Asn Gly Gly Arg Glu His Asp Ser CysAsn 290 295 300 Cys Asp Gly Tyr Thr Asn Ser Ile Tyr Thr Leu Ser Ile SerSer Ala 305 310 315 320 Thr Gln Phe Gly Asn Val Pro Trp Tyr Ser Glu AlaCys Ser Ser Thr 325 330 335 Leu Ala Thr Thr Tyr Ser Ser Gly Asn Gln AsnGlu Lys Gln Ile Val 340 345 350 Thr Thr Asp Leu Arg Gln Lys Cys Thr GluSer His Thr Gly Thr Ser 355 360 365 Ala Ser Ala Pro Leu Ala Ala Gly IleIle Ala Leu Thr Leu Glu Ala 370 375 380 Asn Lys Asn Leu Thr Trp Arg AspMet Gln His Leu Val Val Gln Thr 385 390 395 400 Ser Lys Pro Ala His LeuAsn Ala Asn Asp Trp Ala Thr Asn Gly Val 405 410 415 Gly Arg Lys Val SerHis Ser Tyr Gly Tyr Gly Leu Leu Asp Ala Gly 420 425 430 Ala Met Val AlaLeu Ala Gln Asn Trp Thr Thr Val Ala Pro Gln Arg 435 440 445 Lys Cys IleIle Asp Ile Leu Thr Glu Pro Lys Asp Ile Gly Lys Arg 450 455 460 Leu GluVal Arg Lys Thr Val Thr Ala Cys Leu Gly Glu Pro Asn His 465 470 475 480Ile Thr Arg Leu Glu His Ala Gln Ala Arg Leu Thr Leu Ser Tyr Asn 485 490495 Arg Arg Gly Asp Leu Ala Ile His Leu Val Ser Pro Met Gly Thr Arg 500505 510 Ser Thr Leu Leu Ala Ala Arg Pro His Asp Tyr Ser Ala Asp Gly Phe515 520 525 Asn Asp Trp Ala Phe Met Thr Thr His Ser Trp Asp Glu Asp ProSer 530 535 540 Gly Glu Trp Val Leu Glu Ile Glu Asn Thr Ser Glu Ala AsnAsn Tyr 545 550 555 560 Gly Thr Leu Thr Lys Phe Thr Leu Val Leu Tyr GlyThr Ala Pro Glu 565 570 575 Gly Leu Pro Val Pro Pro Glu Ser Ser Gly CysLys Thr Leu Thr Ser 580 585 590 Ser Gln Ala Cys Val Val Cys Glu Glu GlyPhe Ser Leu His Gln Lys 595 600 605 Ser Cys Val Gln His Cys Pro Pro GlyPhe Ala Pro Gln Val Leu Asp 610 615 620 Thr His Tyr Ser Thr Glu Asn AspVal Glu Thr Ile Arg Ala Ser Val 625 630 635 640 Cys Ala Pro Cys His AlaSer Cys Ala Thr Cys Gln Gly Pro Ala Leu 645 650 655 Thr Asp Cys Leu SerCys Pro Ser His Ala Ser Leu Asp Pro Val Glu 660 665 670 Gln Thr Cys SerArg Gln Ser Gln Ser Ser Arg Glu Ser Pro Pro Gln 675 680 685 Gln Gln ProPro Arg Leu Pro Pro Glu Val Glu Ala Gly Gln Arg Leu 690 695 700 Arg AlaGly Leu Leu Pro Ser His Leu Pro Glu Val Val Ala Gly Leu 705 710 715 720Ser Cys Ala Phe Ile Val Leu Val Phe Val Thr Val Phe Leu Val Leu 725 730735 Gln Leu Arg Ser Gly Phe Ser Phe Arg Gly Val Lys Val Tyr Thr Met 740745 750 Asp Arg Gly Leu Ile Ser Tyr Lys Gly Leu Pro Pro Glu Ala Trp Gln755 760 765 Glu Glu Cys Pro Ser Asp Ser Glu Glu Asp Glu Gly Arg Gly GluArg 770 775 780 Thr Ala Phe Ile Lys Asp Gln Ser Ala Leu 785 790 2 5 PRTArtificial Sequence substitution region 2 Glu Ala Met His Ala 1 5 3 5PRT Artificial Sequence substitution region 3 Ala Trp Phe Gln Trp 1 5 420 PRT Artificial Sequence substitution region 4 Ala Gln Met Trp His GluAla Met Glu Phe Trp Ala Met Gln Phe Glu 1 5 10 15 Ala Met His Ala 20 510 PRT Artificial Sequence substitution region 5 Ala Glu Met Trp His GlnAla Met Glu Val 1 5 10 6 5 PRT Artificial Sequence substitution region 6Ser Tyr Asn Pro Gly 1 5 7 5 PRT Artificial Sequence substitution region7 Ser Tyr Gln Pro Asp 1 5 8 10 PRT Artificial Sequence substitutionregion 8 Gly Ser Pro Tyr Gln Thr Asn Gly Pro Ser 1 5 10 9 10 PRTArtificial Sequence substitution region 9 Gly Ser Pro Asn Ser Gln ProTyr Asp Gly 1 5 10 10 22 DNA Artificial Sequence primer 10 gggggatccctctggcgagt gg 22 11 21 DNA Artificial Sequence primer 11 cggggactctgcgctgctct g 21 12 21 DNA Artificial Sequence primer 12 cagagcagcgcagagtcccc g 21 13 21 DNA Artificial Sequence primer 13 gggggatccccgcggcctag g 21 14 45 DNA Artificial Sequence primer 14 caggccatggaggtgcacct gcctgaggtg gtggccggcc tcagc 45 15 45 DNA Artificial Sequenceprimer 15 gtgccacatc tcggccccct caggggcggt gccatagagt acgag 45 16 45 DNAArtificial Sequence primer 16 cagccctacg acggccacct gcctgaggtggtggccggcc tcagc 45 17 45 DNA Artificial Sequence primer 17 gctgttggggctgcccccct caggggcggt gccatagagt acgag 45 18 21 DNA Artificial Sequenceprimer 18 cacctgcctg aggtggtggc c 21 19 20 DNA Artificial Sequenceprimer 19 cccctcaggg gcggtgccat 20 20 10 PRT Artificial Sequenceinsertion 20 Ala Glu Met Trp His Gln Ala Met Glu Val 1 5 10 21 10 PRTArtificial Sequence insertion 21 Gly Ser Pro Asn Ser Gln Pro Tyr Asp Gly1 5 10

We claim:
 1. A furin polypeptide comprising amino acids, said aminoacids having a sequence which comprises a modification compared to theamino acid sequence of wild-type furin as set forth in SEQ ID NO:1,wherein said modification is present between amino acids Ala 557 andLeu713 inclusive, of wild-type furin, and further wherein saidmodification results in the formation of a loop or alpha-helix structurebetween amino acids Ala 557 and Leu713 of wild-type furin.
 2. A furinpolypeptide according to claim 1 wherein said modification is betweenamino acids 577 and
 713. 3. A furin polypeptide according to claim 1wherein said modification is a substitution of from 5 to 30 amino acids.4. A furin polypeptide according to claim 1 wherein said modification isa substitution of 10 or more amino acids that results in the formationof a helix or loop structure within the mutant furin polypeptide.
 5. Afurin polypeptide according to claim 1 wherein said modificationcomprises adding or substituting amino acids at a furin amino acidposition, wherein the added or substituted amino acids at the positionare selected from the group consisting of alanine (A), leucine (L),phenylalanine (F), tryptophan (W), methionine (M), histidine (H),glutamine (Q) and valine (V).
 6. A furin polypeptide according to claim1 wherein said modification comprises adding or substituting amino acidsat a furin amino acid position, wherein the added or substituted aminoacids at the position are selected from the group consisting of serine(S), isoleucine (I), threonine (T), glutamic acid (E), aspartic acid(D), lysine (K), arginine(R), glycine (G), tyrosine (Y), cysteine(C),asparagine (N), glutamine (Q), proline (P) and hydroxyproline.
 7. Afurin polypeptide according to claim 1 wherein amino acids 578 to 711are deleted.
 8. A furin polypeptide according to claim 1 wherein theamino acids between amino acids 577 and 713 are replaced by amino acidscomprising the sequence AEMWHQAMEV.
 9. A furin polypeptide according toclaim 1 wherein the amino acids between amino acids 577 and 713 arereplaced by amino acids comprising the sequence GSPNSQPYDG.
 10. A furinpolypeptide according to claim 1 wherein said modification is at Arg683.11. A recombinant DNA molecule encoding a furin polypeptide according toclaim
 1. 12. A recombinant expression vector comprising a DNA moleculeaccording to claim 11 operably linked to a heterologous expressioncontrol sequence permitting expression of said furin polypeptide.
 13. Ahost cell comprising a recombinant DNA expression vector according toclaim
 12. 14. A transformed host cell according to claim 13 whichadditionally comprises a polynucleotide encoding at least onerecombinantly expressed precursor polypeptide, wherein said polypeptideis a substrate for the encoded furin polypeptide.
 15. A method for theproduction of a furin polypeptide according to claim 1, said methodcomprising: (a) growing in a nutrient medium a host cell comprising anexpression vector, said expression vector comprising, in order in thedirection of transcription: a transcriptional regulatory region and atranslational initiation region which is functional in said host cell, aDNA sequence encoding a mutant furin polypeptide according to claim 1,and translational and transcriptional termination regions functional insaid host cell, wherein expression of said DNA sequence is regulated bysaid initiation and termination regions; (b) measuring the secretionrate of furin polypeptides with proteolytic activity; and (c) isolatinghost cells expressing furin polypeptides showing reduced secretioncompared to host cells expressing wild type furin.